Determining the variety and ages of 5th instar silkworms for a functional food production through color, phytochemicals, antioxidant activities, absorbance spectra, and gene expression analysis

HongJam (HJ) is a natural food made by steaming mature silkworms, known for its health benefits. The correct type and age of silkworms are essential for its production. This study established standard operating protocols to identify silkworm varieties and ages for making steamed and freeze-dried silkworm powders (SSP) or HJ. By comparing the color of powders made from three silkworm types—White Jade (WJ), Golden-silk (GS), and Juhwang-silk—significant differences in color measurements (ΔE_*ab values) were found. Silkworms of WJ and GS varieties were harvested daily from the 3rd day of the 5th instar stage to maturity, and the color of their powders was analyzed. The color differences (ΔE*ab) were smallest on the 7th day for GS-SSP and the 9th day for WJ-SSP compared to their HJ counterparts. The ΔE_*ab values were the lowest on the GS-SSP produced with the 7th day of the 5th instar silkworm and the WJ-SSP produced with 9th day of the 5th instar silkworm when compared to GS- or WJ-HJ, respectively. The age of silkworms for GS-SSP/HJ production was further determined by analyzing antioxidant activity and the levels of specific plant-based compounds in their extracts. Age identification was also possible by measuring light absorption (200–800 nm) of the extracts and by studying the expression of a gene associated with mature silkworms. In summary, this study offers practical methods to determine the variety and age of silkworms for HJ production, supporting its quality control as a functional food.